High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase

Dinh Thi Quyen, Claudia Schmidt-Dannert, Rolf D. Schmid

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZαA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPα-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4 L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000 U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000 U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter λPL, yielding 600 U/g or 54,000 U/g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents.

Original languageEnglish (US)
Pages (from-to)102-110
Number of pages9
JournalProtein Expression and Purification
Volume28
Issue number1
DOIs
StatePublished - Mar 1 2003
Externally publishedYes

Bibliographical note

Funding Information:
Dinh Thi Quyen gratefully acknowledges a scholarship from the Konrad Adenauer Foundation, Germany.

Keywords

  • Bacillus thermocatenulatus
  • Expression
  • Lipase
  • Pichia pastoris
  • Properties

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